Differential Genes Analysis and Validation of Disulfideptosis in Rat Myocardial Ischemia-Reperfusion Injury Model

Summary

The protocol describes the identification of several novel disulfideptosis-related differential genes associated with myocardial ischemia-reperfusion injury by bioinformatics analysis and experimental validation.

Abstract

Myocardial ischemia-reperfusion injury (MIRI) is an additional injury that occurs during the process of restoring heart tissue blood flow after ischemia-induced injury. MIRI seriously affects the efficacy and short-term and long-term prognosis of reperfusion after myocardial infarction. At present, the mechanism of MIRI is not fully clear. Disulfideptosis is a novel mode of cell death, and the relationship between MIRI and disulfideptosis-related genes (DRGs) expression is still unclear. Firstly, this study explores the differentially expressed genes associated with disulfideptosis in MIRI through bioinformatics analysis. Secondly, by constructing a rat model of MIRI, DRGs were further detected. This study identified 12 related genes, including Myh9, SLC7A11, SLC3A2, Myh7b, ACTB, FLNB, Actn1, Actn4, Flnc, Dbn1 and Pdlim1. Myocardial tissue of rats with MIRI shows obvious pathological damage and apoptosis events. The results of immunohistochemistry indicated that MIRI stimulation increased the expression of GLUT1 protein in myocardial tissue but restricted the expression of F-actin protein. In addition, significant differences in the expression of three proteins were validated using external datasets and MIRI rat models. This study demonstrated that DRGs had significant predictive value in MIRI, providing new prospects for exploring biomarkers and potential therapeutic targets of MIRI.

Introduction

Acute myocardial infarction (AMI) is a severe cardiovascular condition and remains a leading global cause of mortality. Percutaneous coronary intervention has significantly reduced mortality rates in AMI patients¹. However, reperfusion therapy aimed at restoring myocardial blood supply is accompanied by a series of adverse pathological and physiological responses. These processes can result in an increased infarct size, myocardial cell death, sustained ventricular arrhythmias, and sudden death². Myocardial ischemia-reperfusion injury (MIRI) is a complex cardiovascular condition influenced by factors such as cytokines, ch....

Protocol

For this study, nine Sprague-Dawley (SD) male rats, aged 6-8 weeks and weighing 180-220 g, were selected from the Hubei Experimental Animal Research Center [SCXK (Hubei) 20200018]. Rats were kept in specific pathogen free animal houses to acclimatize for 1 week, with a 12 h/12 h light and dark cycle, free drinking and eating. The current study was conducted with approval from the Animal Ethics Committee of The Third Affiliated Hospital of Zunyi Medical University (approval number: (2016)-1-56). All procedures were performed in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of....

Results

Screening of DRGs in MIRI
The GSE214122 dataset from Gene Expression Omnibus included three sham and three MIRI samples data. Using the DESeq2 package in R, 1233 differentially expressed genes (DEGs) were identified between MIRI and sham samples. Based on |log2FC|>2 and FDR<0.05, 417 significantly different genes were further selected using R's pheatmap package (Figure 1A). Then, 15 intersection genes between these 1233 DEGs and 106 DRGs were presented in

Discussion

Disulfideptosis is closely associated with the actin cytoskeleton, a critical cellular structure essential for maintaining cell shape and viability. Composed of actin filaments, the actin cytoskeleton imparts overall cellular shape and structure. F-actin serves as a marker for the cellular cytoskeleton, and under conditions of glucose starvation, disulfide bonds increase significantly, leading to downregulation of F-actin. This phenomenon primarily affects processes and pathways related to the actin cytoskeleton and cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
75% alcohol	Hunan Tongruijian Pharmaceutical Co. Ltd., Hunan, China	85026
6-0 nylon suture	Shanghai Pudong Jinhuan Medical Supplies Co. Ltd., Shanghai, China	CS002
BCA protein quantification kit	Beyotime Biotechnology, Shanghai, China	P0011
Bull serum albumin	Beyotime Biotechnology, Shanghai, China	ST2254
Centrifuge	Hunan Kaida Scientific Instrument Co. Ltd., Hunan, China	KH19A
DAB horseradish peroxidase color development kit	Beyotime Biotechnology, Shanghai, China	P0203
DAPI staining solution	Beyotime Biotechnology, Shanghai, China	C1006
DESeq2 package		Version 4.1
Electric razor	Kelmerpp	235376
Enhanced endogenous peroxidase blocking buffer	Beyotime Biotechnology, Shanghai, China	P0100B
Filter paper	Nanjing Keruicai Equipment Co., Ltd., Nanjing, China	1.00049E+11
Fluorescence microscope	Nikon	ECLIPSE Ci
GEO database	https://www.ncbi.nlm.nih.gov/geo/
Glass grinder	Shanghai Leigu Instrument Co. Ltd., Shanghai, China	B-013002
GraphPad Prism	GraphPad Software	V8.0
Hematoxylin and eosin staining kit	Beyotime Biotechnology, Shanghai, China	C0105M
Image J software	National Institutes of Health, Bethesda, USA	v1.8.0
Improved citrate antigen retrieval solution (50X)	Beyotime Biotechnology, Shanghai, China	P0083
Iodophor	Folca, Shenzhen, China	1.00077E+11
Optical microscope	Nikon	ECLIPSE Ci
Phosphate buffer solution	Beyotime Biotechnology, Shanghai, China	C0221A
Primary antibodies against GLUT1	Proteintech Group, Inc, Wuhan, China	21829-1-AP
Primary antibodies against MYH9	Proteintech Group, Inc, Wuhan, China	11128-1-AP
Primary antibodies against SLC3A2	Wuhan Lingsi Biotechnology Co., Ltd., Wuhan, China	LJS-D-7468
Primary antibodies against SLC7A11	Proteintech Group, Inc, Wuhan, China	26864-1-AP
Protein visualization instrument	Thermo Fisher Scientific Inc.	iBright CL750
Rat board	Zhengzhou Haopai Biotechnology Co. Ltd., Zhengzhou, China	JPB-E
Scalpel	Shanghai Lianhui Medical Supplies Co., Ltd., Shanghai, China	1.00471E+13
Secondary antibody	Wuhan Boster Biological Technology, Ltd., Wuhan, China	BA1054
STRING database	https://cn.string-db.org/	Version 12.0
Triton X-100	Beyotime Biotechnology, Shanghai, China	ST1722
TUNEL apoptosis detection kit	Proteintech Group, Inc., Wuhan, China	PF00009